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FEBS

17734

FEBS Letters

397 (1996) 197-200

The intracellular cytoplasmic domain of the Alzheimers disease amyloid


precursor protein interacts with phosphotyrosine-binding
domain
proteins in the yeast two-hybrid system
Declan M. McLaughlin?, Christopher

C.J. Millera)b?*

BDepartment of Neurology, The Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF, UK
Departments of Neuroscience and Psychology, The Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF, UK
Department of Old Age Psychiatry, The Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF, UK
Received

3 September

1996; revised version

Abstract We have used the yeast two-hybrid system to screen


for proteins that interact with the carboxy-terminal domain of
APP. Six different clones were isolated and sequence analyses
revealed that they encoded domains of a previously described
neuronal protein Fe65, a homologue of Fe65 and a homologue of
protein X11. All of these proteins contain one or more
phosphotyrosine binding (PTB) domains. PTB domain proteins
bind to the sequence Asn-Pro-X-Tyr when the Tyr is phosphorylated and are believed to function in signal transduction. APP
contains such a motif. These results are consistent with a role for
APP in signal transduction mechanisms.
Key words: APP; Alzheimers
disease;
Phosphotyrosine-binding
domain

Fe65;

X11;

1. Introduction
Deposits of P-amyloid in neuritic plaques and blood vessel
walls are a principal pathological
feature in the brains of
patients
with Alzheimers
disease. These amyloid deposits
contain the 3943 amino acid P-amyloid
peptide which is
derived by proteolytic
cleavage from its much larger precursor, the amyloid precursor protein (APP). P-amyloid is a normal physiological
product of APP metabolism.
However, it is
thought that aberrant processing
of APP to produce either
increased
amounts
of P-amyloid
or longer (42/43 amino
acid) and hence more amyloidogenic
P-amyloid isoforms, is
a primary pathogenic
event leading to amyloid deposition in
Alzheimers disease (see [l-3] for reviews).
APP gene mRNA transcripts are alternatively spliced so as
to generate a number of different APP molecules; the principal isoforms comprising
69.5, 751 and 770 amino acids. The
longer isoforms contain a domain that displays homology to
the Kunitz class of protease inhibitors [4]. APP695 is the predominant isoform found in brain.
APP is a membrane-spanning
protein which crosses the
plasma membrane once and contains a large extracellular domain (which in APP751 and APP770 contains the Kunitz
protease inhibitor domain) and a smaller 47 amino acid intracellular domain. P-amyloid is derived from sequences contained within and just external to the membrane
spanning
domain (see [l-3] for reviews).
Although secreted extracellular domains of APP containing

*Corresponding
author. Department
of Neuroscience,
The Institute of
Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF, UK.
Fax: (44) 171-708-0017.
0014-5793/96/$12.00
PIISOOl4-5793(96)01

0 1996 Federation
128-3

of European

Biochemical

Societies.

received

10 September

1996

the Kunitz protease inhibitor domain may act as extracellular


serine protease inhibitors,
[5,6] the function of membranebound APP is not fully understood.
However,
APP has
some structural features of cell surface receptors [7] and the
intracellular
domain of APP has been shown to bind G, [8]
which suggests that it may be involved in signal transduction
processes. In common with a number of cell surface receptors,
membrane-bound
APP has been shown to be re-internalised
into lysosomes where intact P-amyloid containing
fragments
are produced
and this is believed to be one route for the
production
of P-amyloid [9-121. The carboxy terminal intracellular domain of APP contains the motif Asn-Pro-Thr-Tyr
which is a sequence known to mediate re-internalisation
via
clathrin-coated
pits [13]. If the tyrosine in this sequence is
phosphorylated,
then this motif is also a consensus sequence
for binding to phosphotyrosine
binding (PTB) domains [14
161 (and see [17] for review). To understand
further the function of APP and the mechanism(s)
of P-amyloid production,
we have used the yeast two-hybrid system to identify proteins
that interact with the intracellular carboxy-terminal
portion of
APP. Here we demonstrate
that this domain of APP interacts
with a number of PTB domain containing proteins.
2. Materials and methods
To generate a GAL-4 DNA binding domain-APP
fusion bait plasmid, sequences encoding the intracellular
carboxy-terminal
47 amino
acids of APP (APPc) were amplified by PCR and cloned into the
GAL-4 DNA binding domain yeast shuttle vector pY1 [18] so as to
produce pY1APPc. Clones were sequenced to check that no PCR
errors had been introduced
and that APP sequences were in-frame
with GAL-4 encoding sequences. pY 1APPc was used to screen a human brain cDNA library containing
the GAL-4 transactivation
domain in pGADl0
(Clontech) by co-transformation
of both plasmids
into yeast Yl90. Yeast transformations
were performed using lithium
acetate/heat
shock as described (Clontech). Y 190 possesses both IacZ
and His GAL reporters.
Transformants
were grown for 8 days at
30C on synthetic selection medium lacking tryptophan,
leucine and
histidine and containing
25 mM 3-amino triazole. Large (> 2 mm)
colonies surviving selection were picked and assayed for B-galactosidase activity using a freeze-fracture
assay. Plasmids from colonies
positive on both the nutrient (His) and B-galactosidase
assays were
rescued into Escherichia coli HBlOl. To check that positive plasmids
only transactivated
in the presence of pY IAPPc, yeast Yl90 were retransformed
with the candidate plasmids either alone, or with pY 1 or
pLAM5 (Clontech) that contains the GAL-4 DNA binding domain
fused to an extraneous
fragment of laminin and colonies again assayed for B-galactosidase
activity. Positive controls included transformation of Yl90 with pCL1 (that contains full-length GAL-4) and cotransformation
with pVA3 and pTD1 (that contain respectively the
GAL-4 DNA binding domain fused to p53 encoding sequences, and
GAL-4 transactivation
domain fused to SV40 large T antigen encoding sequences). pCL1, pVA3 and pTD1 were obtained from Clontech.
All rights

reserved.

D. M. McLaughlin,

198
Plasmids from positive clones which transactivated only in the presence of pYlAPPc were sequenced and homology searches performed
using the BLAST network service.

C. C. J. Miller I FEBS Letters

397 (1996)

197-200

encoding 441 amino acids that bore greatest similarity to a


mouse homologue of protein X11 [20] (99% homology at the
amino acid sequence level) but which displayed less homology
to human protein Xl1 [21] (83% homology at the amino acid
sequence level). Further searches revealed that overlapping
but identical sequences to the clone 6 insert had previously
been deposited in the database (Accession numbers R89683,
Rl3101, R18654 and T16098). Clone 6 contains an in-frame
stop codon but no in-frame ATG with surrounding
Kozak
sequence and so may well code for a truncated carboxy-terminal fragment. To simplify the terminology in this report, we
have termed the protein encoded by clone 6, protein X1 l-like.
Analyses of the proteins encoded by the six clones revealed
that they all contain at least one phosphotyrosine
binding
(PTB) domain. The HFe65 clones contain two PTB domains;
the HFe65-like clone contains one PTB domain (indeed, the
amino acid sequences encoded in the clone represent a single
PTB domain);
and the protein X11-like clone contains one
PTB domain. The PTB domains of HFe65, HFe65-like and
protein X11-like are shown and aligned in Figs. 1 and 2. It
has previously been noted that both Fe65 and protein Xl1
have PTB domains [22,23].
Fiore et al. [23] have shown that the rat Fe65 PTB domains

3. Results and discussion


Approximately
10 independent
library
colonies
were
screened with pYlAPPc
using the yeast two-hybrid
system
and six positive clones identified. Analyses of the open reading
frames of these clones revealed that threecontained
identical
inserts which encoded the carboxy-terminal
422 amino acids
of the human homologue
(HFe65) of a previously described
rat neuronal protein, Fe65 [19]. A fourth clone encoded the
carboxy-terminal
648 amino acids of HFe6.5. The fifth clone
contained sequences that encoded an open reading frame of
150 amino acids that were 64% identical to HFe65 at the
amino acid sequence level. This suggests that clone 5 might
contain a homologue
of HFe65 (HFe65-like) and that there
may be a family of Fe65 proteins. No in-frame start codon
with surrounding
Kozak sequence or stop codon were present
in the clone 5 insert which indicates that it lacks both the
amino- and carboxy-terminal
encoding DNA sequences of
this protein. The sixth clone contained an open reading frame

A
Fe65/rat

(NJ

143 NPGIKCFAVRSLGWVEMTEEELAPGRSSVAVNNCIRQLSYHKNNLHDPMS
IIIIIIIIIIIIIIIIlIlIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

Fe65/human
Fe65/rat

(N)
(NJ

NPGIKCFAVRSLGWVEMTEEELAPGRSSVAVNNCIRQLSYHKNNLHDPMS
GGWGEGKDLLLQLEDETLKLVEPQNQTLLHAQPIVSIR~GVGRDSGRERDFAY
IIIIIIIIIIIIIIIlIIIIIIII

Fe65/human
Fe65/rat

(NJ

(N)

1IIIIIl

1111111111111111111

GGWGEGKDLLLQLEDETLKLVEPQSQALLHAQPIISIRVWGVGRDSGRERDFAY
VARDKLTQMLKCHVFRCEAPAKNIATSLHEICSKIMSERRNA
lIIIIIIlIIIIIIIIIIIIIIIIlIIIIIIIIIII

Fe65/human

(NJ

288

x04689

IIIII

VARDKLTQMLKCHVFRCEAPAKNIATSLHEICSKIMAERRNA

B
Fe65/rat

(C)

326 NELVQKFQVYYLGNVPVAKPVGVDVINGALESVLSSSSREQWTPSHVSVA
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

Fe65/human

NELVQKFQVYYLGNVPVAKPVGVDVINGALESVLSSSSREQWTPSHVSVA

(Cl

III

Fe65-like
Fe65/rat

(Cl
(Cl

III

II

IIII

II

I I

IIIIIIIIIIIIIIIIlIIlllllllllllllllllllllllllll

PATLTIL-HQQTEAVLGECRVRFLSFLSFLAVGRD~TFAFI~GPASFCC~FWC
II

Fe65-like
Fe65/rat

PATLTIL-HQQTEAVLGECRVRFLSFLSFLAVGRD~TFAFI~GPASFCC~FWC
Illllll

Fe65/human

III

TELVQKFHVQYLGMLPVDKPVGMDILNSAIENLMTSSNKEDWLSVNMNVA

II

IIIIIIIII

II

IIIIIIIII

II

III

DATVTVISEKNEEEVLVECRVRFLSFMGVGKDVHTFAFIM
(Cl

EPNAASLSEAVQAACMLRYQKCLDA

454

x04689

I I I I I I I I I I I I I I I I II I II I I I I

Fe65/human

(Cl

EPNAASLSEAVQAACMLRYQKCLDA
I I I I

Fe65-like

IIIIIIIIIIIIIIIl

EPNAGNVSEAVQAACMLRYQKCLVA

Fig. 1. Alignments of the PTB domain amino acid sequences from rat and human Fe65 and human Fe65-like protein.
amino-terminal
(N) PTB domains of human and rat Pe65.
with the PTB domain identified in human Fe654ke
protein.
vidual proteins are listed at the end of relevant sequences.

A: Alignment
of the
B: Alignment of the carboxy-terminal
(C) PTB domains of rat and human Fe65
Dashed line indicates a gap in the alignment. The accession numbers for the indi-

D. M. MeLoughlin,

C. C. J. Miller1 FEBS Letters

X11-like

397 (I 996) 197-200

199

LIDGIIFAANYLGSTQLLSERNPSKNIRMMQAQEAVSRVKRMQKAAKIKK
IIIlIIIlIIIIIIIIIlIII

IIIIIIIIIIIII

IIIIIIIIIIIIII

Xll/mouse

298

LIDGIIFAANYLGSTQLLSERTPSKNIRMMQAQEAASRVKRMQ~KIKK

x11

326

LIDGIIFAANYLGSTQLLSDKTPSKNVRMMQAQEAVSRIKMAQKLAKSRK

X11-like

KANSEGDAQTLTEVDLFISTQRIKVLNADTQETMMDHALRTISYIADIGNIWLM

Xll/mouse

KANSEGDAQTLTEVDLFISTQRIKVLNADTQETMMDHALRTISYIADIGNIWLM

x11

KA-PEGESQPMTEVDLFILTQRIKVLNADTQETMMDHPLRIGNIWLM

X11-like

ARRRMPRSASQDCIETTPGAQEGKKQYKMICHVFESEDAQLIAQSIGQAFSVAYQ

Xll/mouse

ARRRMPRSASQDCIETTPGAQEGKKQYKMICHVFESEDAQLIAQSIGQAFSVAYQ

x11

ARRRIPRSNSQENVEASHPSQDGKRQYKMICHVFESEDAQLIAQSIGQAFSVAYQ

X11-like

EFLRA

Xll/mouse

EFLRA

I I I I I I I I I I I I I I I I I I I

IIIII

IIIIIIII

II

II

II

IIIIIIIIIIIIIIIIIIIllllllllllllllIIIIIIIIIIIIIIIIIIIIII
II

II

IIIIIII

IIlIIIIIlIIIIIIIII

IIIIIIIlIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
III1

III

II

II

IIIIIIIIIIIIIIIIIIIIIIIIIIIIII

I I I I I

462

L34676

489

LO4953

I I I I I
x11

EFLRA

Fig. 2. Alignments of the PTB domain sectuences from the amino acid sequences of the mouse and human versions of the Xl 1 protein plus the
PT-B domain sequence of the human Xl I-iike protein.

interact with the carboxy-terminus


of APP in the yeast twohybrid system and in co-precipitation
experiments
with Glutathione-S
transferase-Fe65
fusion proteins.
However, their
approach involved utilising Fe65 sequences as bait to screen
a library rather than APP sequences. Our findings that the
intracellular
domain
of APP interacts
with human
Fe65
when APP is used as bait thus confirms and extends their
findings.
In addition, we demonstrate
that two other PTB domain
proteins, HFe65-like (a homologue of Fe65) and protein Xl llike (a homologue of protein Xl l), all interact with the intracellular carboxy-terminal
domain of APP in the yeast twohybrid system. Protein Xl 1 was originally identified as a candidate for the Friedreichs ataxia disease gene [21]. A mouse
homologue
of protein X11 has been cloned [20] and the sequences we have isolated for protein Xl l-like protein display
high and greater homology to this murine homologue than to
protein X11 (protein X11-like is 99% identical to the mouse
protein X11 but 83% identical to human protein X11) which
suggests that there might be a family of these proteins. Thus
both the mouse and human genomes contain a protein Xl llike gene. In situ hybridisation
studies with mouse protein
Xl l-like probes reveal that its transcripts are widely expressed
throughout
the nervous system including the cortex and hippocampus,
two of the regions affected in Alzheimers disease

Pll.
The Fe65-like sequences we have isolated encode for 150
amino acids and this appears to essentially comprise a single
PTB domain (see [22] for alignment of some PTB domains).
Thus, it appears that an isolated PTB domain from Fe65-like
protein is sufficient for binding to the carboxy-terminal
intracellular domain of APP.
The PTB domain was originally identified in the Src homology 2 (SH2) containing
protein She [24,25]. Like SH2 do-

mains, PTB domains appear to bind tyrosine phosphorylated


proteins although SH2 and PTB domains are structurally unrelated. It is believed that PTB domains impart on proteins an
ability to bind the sequence motif Asn-Pro-X-Tyr
when the
Tyr is phosphorylated
and that PTB domain containing proteins function in mechanisms for transducing
signals from the
cell surface [15,17,24,25]. The intracellular
portion of APP
used here as bait contains one Asn-Pro-X-Tyr
motif.
APP has previously been shown to interact with G, and to
have cell signalling functions in vitro via its carboxy-terminal
sequences [8,26]. In addition, APP has recently been reported
to interact with N-Pak, a neural-specific
p21 activated kinase
[27]. Such observations
suggest that APP may have a role in
signal transduction.
In this context, it is striking that we find
that three different PTB domain containing
proteins will interact with APP in the yeast two-hybrid system; proteins containing PTB domains are believed to function in signal transduction [17]. Thus our findings further implicate APP (either
directly or indirectly) in such cell signalling mechanisms.
The Asn-Pro-X-Tyr
motif is also believed to mediate reinternalisation
of receptors from the cell surface via clathrincoated pits [13]. Such a re-internalisation
of APP into lysosomes is one route for the generation of secreted P-amyloid
and mutation of the Asn-Pro-X-Tyr
motif is inhibitory to pamyloid production
[12]. In this respect, it will be interesting
to determine how expression of the three PTB domain proteins described here influence APP processing and b-amyloid
production.
Such studies are currently underway in this laboratory.
Acknowledgements:
This work was supported
by grants from the
Wellcome Trust and MRC to C.C.J.M. D.M. is supported by a UK
Alzheimers Disease Society fellowship. We thank Dr. Peter Broad,
Zeneca Pharmaceuticals
for the gift of pY plasmids and for much
helpful advice on the two hybrid system.

200

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